The invention relates to an arrangement for surface plasmon resonance spectroscopy, which in particular can be used for a direct analysis of the interaction between bio-molecules and which permits a multi-component analysis.
There is known a very sensitive method for specifying the characteristics of boundary faces that, in the references, is referred to as surface plasmon resonance spectroscopy, generally designated as SPR (surface plasmon resonance). This method is based upon the optical excitation of surface plasmons in thin metal layers. The resonance conditions for the excitation of surface plasmons strongly depend on the optical properties of the dielectrics surrounding the metal layer. Hence, it is principally feasible to determine the refractive index and the layer thickness of thin dielectric layers with high precision.
SPR-spectroscopy finds an increasing application in the biochemical analysis, since it permits a direct analysis of the interactions between the bio-molecules (for example, antibody/antigen reactions). To this end a reactant (ligand) is immobilized on the metal surface, the other reactant (analyt) is passed over the surface in solution. The interaction can be directly detected as an increase in layer thickness; there is no marking of the reactants necessary as, for example, with the radioimmunoassay (RIA) or the enzym-linked immunosorbent assay (ELISA). These and further methods of the prior art have been described in detail by Striebel, Ch.; Brecht, A.; Gauglitz, G. in Biosensors and Bioelectronics 9 (1994), 139-146. Nearest to the present invention comes the SPR-method, described therein, in which light originating from a light conducting fiber is directed via a collimator and an optical polarizer upon an optical prism, on the base of which a thin silver coat enabling SPR is deposited that is covered by a chip, which is provided with a sample flow channel extending perpendicularly to the illumination propagation direction. The light that is reflected at and affected by the silver coat in response to the respective surface coverage, is then directed to a diode array spectrometer via a light conducting fiber. A simultaneous multi-component analysis is not possible with the arrangement described therein, because there can only be provided one optical and one fluid channel. In WO 97/40366 an arrangement is described, in which the detection of a plurality of samples arranged on a substrate sheet is realized in that the beams reflected on the samples are simultaneously imaged upon a matrix-shaped detector (CCD-matrix or video camera). All samples are successively illuminated with light of different wavelengths in order to determine the resonance wavelength. A tunable light source or a scanning monochromator is provided in the emitted path of rays for the selection of the wavelengths. Though the spectral measurement avoids the disadvantages of the intensity measurement described herein below, this system, however, can only be realized under considerable costs and only with comparatively large stationary devices, due to the required components.
Furthermore, arrangements of the prior art are known, which make use of the SPR method, but only provide for a pure angular detection of the totally reflected light. So a device is known from the product specification of the firm Biacore AB, Rapsgatan 7, S-75450 Uppsala, Sweden 1996, the setup of which in principle corresponds to the above described arrangement, whereby the illumination is provided by converging light beams and as a detecting element a diode array is directly and as a structural unit associated to the second lens system. Such a setup requires a comparatively large and mechanically bulky measuring head that can exclusively be employed in a stationary device. Furthermore, Berger, Ch. E. H. et al in xe2x80x9cSurface plasmon resonance multisensingxe2x80x9d, Anal. Chem. 1998, 70, p. 703-706 describe an arrangement that permits a multi-channel measurement, which however is based upon an intensity measurement as a measuring principle, which is connected with the known disadvantages, namely the high requirements for a stability of the light source and of the detector elements that only can be obtained with considerable expenditures for a control system. Furthermore, the arrangement described therein limits the angular range otherwise basically available, in which plasmon resonance oscillations can be detected, since only a narrow angular range can actually be exploited by the measuring method described there. Moreover, the arrangement described there and which makes use of a video camera and of a video recording, has comparatively high expenditures for equipment, apart from the fact that a simultaneous multi-channel measurement is not provided for, since the measuring results can only be evaluated afterwards in time sequence.
A further arrangement belonging to that group of arrangements is described by Brink, G. et al, in xe2x80x9cNear infrared surface plasmon resonance in silicon-based sensorxe2x80x9d, Sensors and Actuators B 24-25, 1995, p. 756-761. Therein a silicon wafer is used for the sample chip, the silicon wafer is provided with a step that is differently coated so that two resonant angular ranges and, hence, two channels can be detected, when the illumination spots covers both faces of the step. Even when using a plurality of steps therein, the number of exploitable channels would be principally limited by the angular range, in which the SPR method will work. Still more serious will be the disadvantage that with the different spectral position of the steps, the mutual sensitivity of the steps will not be identical.
Due to the problems mentioned, the commercially available SPR measuring technique has failed to find a wide application.
It is an object of the invention to provide a miniaturized arrangement for surface plasmon resonance spectroscopy that is designed as a cost-effective and portable unit and that is also adapted to perform a multi-component analysis, in particular for the interaction between bio-molecules.
In the frame of the invention, light originating from a broad-band light source and directed via a light conducting fiber to a collimator of a comparatively wide exit aperture is, by virtue of said collimator, directed upon an optical prism, at the base face of which a sample receiving cell is located, the bottom of which is provided with a thin metal layer enabling the SPR-method. It lies within the scope of the invention to also provide the base face of said prism with the metal layer mentioned or, by way of immersion, to deposit a metal coated substrate on the base face of the prism. The very essence of the invention consists in providing a multifold and in different positions switchable diaphragm between the collimator and the first prism entrance face, whereby in each switching state of the diaphragm definite ranges of the bottom of the sample cell are illuminated. The respectively affected and reflected light is fed, via a second collimator with connected light conducting fiber, into a polychromator, the spectral signals of which are captured by a CCD-array or by a diode array and passed on to an evaluation and control unit, in which a correlation is performed to both, the respective switching states of the diaphragm and to a signal obtained from a reference channel provided on the sample cell.